Stimulation of pancreatic islets with glucose results in phospholipid hydrolysis and accumulation of non-esterified arachidonic acids, as demonstrated by isotope dilution mass spectrometry. Pancreatic islets express a Ca2+-independent phospholipase A2 (CaI-PLA2) activity which is sensitive to inhibition by a haloenol lactone suicide substrate (HELSS) that also attenuates glucose-induced hydrolysis of arachidonic acid from islet phospholipids and insulin secretion. A cDNA has been cloned from a rat islet cDNA library which encodes a protein with a deduced amino acid sequence of 751 residues that is homologous to a CaI-PLA2 enzyme recently cloned from CHO cells. Transient transfection of both COS-7 cells and CHO cells with the cloned islet CaI-PLA2 cDNA resulted in an increase in cellular CaI-PLA2 activity, and this activity was susceptible to inhibition by HELSS. The domain of the islet CaI-PLA2 from amino acid residues 150-414 is composed of 8 stretches of a repeating sequen ce motif of approximately 33 amino acid residues in length which is highly homologous to domains of ankyrin which bind both tubulin and integral membrane proteins, including several proteins which regulate ionic fluxes across membranes. These findings complement previous pharmacologic observations which suggest that CaI-PLA2 may participate in regulating trans-membrane ion flux in glucose-stimulated b-cells.